PROJECT SUMMARY: A major goal of my career as an independent researcher is to understand how integral membrane proteins fold in the crowded environment of the endoplasmic reticulum (ER). Disruptions in the fidelity of this process lead to a number of human diseases including Alzheimer?s, Parkinson?s and Charcot-Marie-Tooth Disease (CMTD). By better understanding this process, I hope to eventually identify novel therapeutic strategies for treating patients afflicted with these diseases. CMTD is the most common neuropathy of the peripheral nervous system (PNS) and afflicts 1 in 2,500 individuals. Over 80% of patients afflicted with this disease have a mutation in the protein encoding PMP22 gene. PMP22 is a tetraspan integral membrane protein that is highly expressed at the plasma membrane (PM) of myelinating Schwann cells of the PNS. Improper gene dosage of PMP22 in CMTD patients results in either too much or too little PMP22 at the PM of Schwann cells. The altered amount of PMP22 at the PM is believed to cause the myelin abnormalities observed in these patients. There are currently no treatments for CMTD. PMP22 folds in the ER under the surveillance of the ER quality control (QC) system. This system both helps PMP22 to fold as well as mediates the ultimate decision whether to traffic PMP22 forward towards the PM or to retain the protein in the ER and ultimately target it for degradation. The goals of this proposal are to understand the factors that mediate this decision process. Previous results show that wild type (WT) PMP22 and PMP22 disease mutants have different protein interactions in the ER QC system leading to varying PM expression. Preliminary data shows that selective modulation of protein expression in ER can alter the cell surface trafficking of PMP22 and its disease mutants. Additionally, PMP22 associates with cholesterol rich membrane domains at the PM. It has been shown for another membrane protein of the PNS, P0, that association with cholesterol in the ER is necessary for cell surface trafficking. Schwann cells derived from PMP22 -/- mice show decreased migration and adhesion capabilities and lower PM cholesterol content compared to WT Schwann cells. This indicates that PMP22 association with cholesterol rich domains is important for Schwann cell function. It is my hypothesis that PMP22 cell surface trafficking can be modulated by both changing its protein interaction network in the ER and through altering its ability to associate with cholesterol rich membrane domains. To address this hypothesis, I will: (1) uncover mutation specific changes to the PMP22 interactome in the ER that modulate its cell surface trafficking and (2) examine the factors that cause PMP22 to associate with cholesterol rich membrane domains and assess the effects of cholesterol association on PMP22 trafficking. The results from this proposal will uncover novel protein interactions and factors that affect PMP22 trafficking and cholesterol association. This information will open novel avenues for scientists attempting to develop therapeutics for patients who suffer from CMTD.